Effects of epidermal growth factor and hormones on granulosa expansion and nuclear maturation of dog oocytes in vitro

Abstract

Gonadotropins, steroids and growth factors stimulate or inhibit cumulus expansion, nuclear maturation, or both, of most mammalian oocytes in vitro. The objective was to evaluate the effects of epidermal growth factor (EGF) and various hormone combinations on in vitro granulosa/cumulus (G-C) expansion and nuclear maturation of domestic dog oocytes derived from advanced preantral and early antral follicles. Follicles were collected after enzymatic digestion of ovarian tissue and cultured for 66 h in F-12/DME with 20% fetal bovine serum, 2 mM glutamine and 1% antibiotic–antimycotic (Control). Treatments comprised the following groups; each was cultured both with and without EGF (50 ng/mL): Control, FSH (0.5 μg/mL), LH (5 μg/mL), estradiol-17β (E2, 1 μg/mL), FSH + LH, and FSH + LH + E2. Granulosa/cumulus expansion was scored on a scale of 0 (no expansion) to +3 (maximum expansion). The interaction between EGF and hormone treatment affected ( P = 0.011) maximum G-C expansion. With the exception of the E2 group, EGF increased ( P < 0.05) the proportion of oocytes exhibiting +3 expansion. The synergism of E2 with FSH + LH enhanced maximum G-C expansion; compared to all other treatments, the greatest expansion was observed in the FSH + LH + E2+EGF group (83.5 ± 3.5%). When cultured in EGF alone, oocytes failed to reach metaphase I-II (MI-MII) stages. The interaction between EGF and hormone treatment tended ( P = 0.089) to increase the proportion of oocytes resuming or completing nuclear maturation (GVBD-MII). In addition, supplementing culture media with hormones increased ( P = 0.010) the GVBD-MII rate. Therefore, EGF in combination with FSH and LH enhanced G-C expansion of cultured canine oocytes, with no significant effect on the proportion of oocytes derived from advanced preantral and early antral follicles that reached MI-MII.